우리는 프랭크 에르빈, 로버타 Palmour 및 영장류 작품들의 지속적인 지원 세인트 키츠, 서인도 제도에있는 Behavioural 과학 재단 연구소의 직원에게 감사하고 있습니다. 보건 연구 (AC)과 국립 공학 및 캐나다 (MP)의 연구위원회의 캐나다 연구소에서 운영 보조금 -이 작품은 깨지기 쉬운 X 리서치 SZ 캐나다의 재단 (FXRFC)와의 화목에 의해 지원되었다.

Immunohistochemisty 다양한 실험 동물 모델의 두뇌에있는 단백질 표현을 특성화를 위해 가장 널리 사용되는 기법 중 하나입니다. 유사한 두뇌 크기와 설치류 및 기타 일반적인 실험 모델의 두뇌에 체계적인 immunohistochemical 절차를 수행하기 위해 상대적으로 쉽습니다. 그러나, 전체 원숭이의 두뇌에 걸쳐 같은 면역 절차를 수행하는 방법에 대한 포괄적인 계정을 제공하는 우리의 지식에 게시된 작업은 없습니다. 어떤 다음은 다양한 대상 단백질의 대규모 immunohistochemical 감지에 대한 전체 원숭이 두뇌를 준비하는 방법에 대한 자세한 설명입니다. 이 작품은 상업 및 학문적 노력 사이의 협력의 결과로 떠오르고있다. 이와 같이 포함하고 sectioning 조직에 관한 세부 사항은 NSA의 독점적인 지식 남아 있습니다. 1 부 : 동물 치료 및 조직 준비 성인 vervet 원숭이 (Cercopithecus aethiops)에서 두뇌는 현재 프로토콜에 사용됩니다. 모든 절차는 생물 의학 연구 1 동물의 사용과 관리에 대한 애니멀 케어의 캐나다 협의회 (CCAC) 지침을 준수 실시하고 있습니다. <ol><li> 동물은 깊이 나트륨 pentobarbital (25 MG / kg, IV)와 완전히 exsanguinated 때까지 0.1 M PBS로 transcardially perfused의 과다로 euthanized 케타민을 하이드로 클로라이드 (10 MG / kg, IM)와 마취입니다. </li><li> 이것은 5 분 (~ 1리터)을 PBS에 4 % paraformaldehyde 솔루션옵니다. </li><li> 두뇌가 컨테이너의 하단에 싱크 ˚ C까지 externalized 두뇌는 0.02 %의 나트륨 azide를 포함한 등급 PBS 버퍼 자당 솔루션 (순서 10, 20, 30 %)에 위치 4에서 관리하고 있습니다. 두뇌가 냉동 sectioning를 겪습 때까지 솔루션은 수일마다 교체됩니다. </li><li> 그들의 독점 MultiBrain에 들어서는 ™ 삽입 및 냉동 sectioning 기술, 두뇌는 신경 과학 어소 (녹스빌, 테네시 NSA)로 전송됩니다. </li><li> 섹션이 제대로 지향되어야 (즉, 왼쪽, 오른쪽 방향)와 histological 처리가 완료된 후 다시 정렬 수 있도록 세븐 (7) 정렬 랜드마크가 포함 행렬에 배치됩니다. </li><li> 각각의 시리얼 부분이 블록에서 수집되기 전에 냉동 블록 디지털 블록 얼​​굴 사진입니다. 디지털 이미지뿐만 아니라이 두뇌의 부분은 나중에 각각 해부 및 histological지도 3D 재건에 사용될 수 있습니다. </li><li> 시리얼 자유 부동 코로나 섹션은 섹션 50 μm의의 두께, 전체 두뇌에서 수집됩니다. </li></ol> 2 부 : Histological 처리 섹션은 주어진 공간 간격으로 선택됩니다 (예, 500 μm의) 우리의 실험 목적을 위해 다음과 같은 항체로 처리되었습니다 연약한 - X 정신 지체 단백질 (FMRP, Chemicon, 테메큘라, CA), SMI32 (Sternberger Monoclonals 주식 회사; 볼티모어, MD), 그리고 NeuN (Chemicon, 테메큘라, CA). FMRP는 다량으로 정상과 순열 사업자 두뇌의 뉴런이 발견 세포질 단백질이다. NeuN (핵의 연결)는 구체적으로 대부분의 뉴런에 존재하며 핵의 연결, perikarya 일부 근위의 연결 프로세스 3 배포되는 DNA 바인딩 신경 세포 특정 단백질 NeuN를 인식합니다. SMI - 32 neurofilament 단백질 4 비 phosphorylated 에피토프을 인식 단클론 항체이다. <ol><li> 500 μm의의 간격, 약 140 섹션은 항체마다 사용됩니다. </li><li> 모든 incubations, 세척 및 얼룩 단계는 전문 얼룩 요리와 바구니 (; 녹스빌, 테네시 HistoTools)를 사용하여 가벼운 동요와 함께 진행하고 있습니다. </li><li> 섹션 먼저 항체 분자의 특이 현상이 자꾸 발생 배경 얼룩을 줄이기 위해 0.1 M PBS/0.3 % 트리톤 X - 100 (TX) / 60 분 5 % 정상 쉰 혈청 (NHS)이있는 솔루션에 incubated 수 있습니다. </li><li> 공급 업체의 지시에 따라, immunostaining FMRP에 대한 표시 섹션 항원 Unmasking 솔루션 (버링게임, CA 벡터 연구소)를 사용하여 항원 복구의 추가 단계를 받고있다. </li><li> 섹션은 다음 하룻밤과 0.1 M PBS/0.3 % TX / 3 % NHS를 (NeuN 항체에 대한 FMRP 및 SMI - 32과 1 / 2, 000에 대한 5분의 1, 000의 희석 요소로 포함하는 각각의 항체 솔루션에서 별도의 일 incubated 수 있습니다. </li><li> 1 /; 다음날은 섹션은 말에서 제기 biotinylated 방지 마우스 이차 항체에 실온에서 2 H (벡터 연구소에 대한 부화 다음 세척 용액 세 10 분 기간 (0.1 M PBS/0.3 % TX)에 씻어 아르 0.1 M PBS/0.3 % TX / 3 % NHS 1,000 희석). </li><li> 3 10 - 분 세척을 추가로 설정 후, 섹션은 실온에서 1 H에 대한 아비딘 - 비오틴 복합 양고추냉이 퍼옥시데이즈 복합 (벡터 연구소)의 솔루션에 배치됩니다. </li><li> 마지막으로, 세척 다른 설정 후, 섹션은 니켈 향상된 디아 10 분 대상이 아르minobenzidine (NI - DAB) 진한 파란색이 immunoreactive 뉴런 이내에 얼룩이 생성 반응. </li><li> histological 절차의 완료에, 부분은 젤라틴 - 코팅 유리 슬라이드에 탑재하고 있습니다. </li><li> 모든 섹션은 공기 건조 하룻밤되고 표준 절차에 따라 coverslipped. </li></ol> 파트 3 : 대표 결과 : 이 방법은 전체 원숭이 두뇌에 걸쳐 관심의 대상 단백질의 완전한 표현 프로필을 생산하고 있습니다. 여기 FMRP, NeuN과 같은 원숭이의 뇌에 SMI32 표현의 스냅샷을 제공하는 대표적인 코로나 섹션을 표시합니다. <img src="/files/ftp_upload/1286/1286fig1.png" alt="" /> 그림 1 : 스테 이닝 요리 (A)와 바구니 (B)가 면역 무료 - 부동 섹션의 대규모 일괄 처리에 사용. <img src="/files/ftp_upload/1286/1286fig2.png" alt="" /> 그림 2 : FMRP (A), NeuN (B)와 SMI32 (C) 항체에 대한 스테인드 vervet 원숭이 머리에서 대표 코로나 섹션.

<ol>
	<li>Olfert, E. D., Cross, B. M., McWilliam, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to the Care and Use of Experimental Animals. , (1993).</li><li>Devys, D., Lutz, Y., Rouyer, N., Bellocq, J. P., Mandel, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FMR-1+protein+is+cytoplasmic,+most+abundant+in+neurons+and+appears+normal+in+carriers+of+a+fragile+X+premutation.">The FMR-1 protein is cytoplasmic, most abundant in neurons and appears normal in carriers of a fragile X premutation.</a> Nat Genet. 4, 335-340 (1993).</li><li>Mullen, R. J., Buck, C. R., Smith, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NeuN,+a+neuronal+specific+nuclear+protein+in+vertebrates.">NeuN, a neuronal specific nuclear protein in vertebrates.</a> Development. 116, 201-211 (1992).</li><li>Sternberger, L. A., Sternberger, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monoclonal+antibodies+distinguish+phosphorylated+and+nonphosphorylated+forms+of+neurofilaments+in+situ.">Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.</a> Proc Natl Acad Sci U S A. 80, 6126-6130 (1983).</li></ol>

The authors have nothing to disclose.

가능한 전체 원숭이의 뇌에 단백질의 대규모 감지하게이 절차에서 두 중요한 단계가 있습니다. 하나는 NSA의 독점적인 지식 남아 삽입과 sectioning 프로토콜입니다. 다른 하나는 HistoTools에서 제공하는 얼룩 요리와 바구니를 사용하는 것입니다. 후자는 주어진 시간에 많은 (~ 40) 섹션을 쉽고 빠르게 처리하실 수 있습니다. 또한 균일하게 뇌 전체 부분을 치료를위한 수단을 제공하고 과학적으로 소리 histological 치료를합니다. 또한, 임베디드 랜드마크의 사용은 디지털 말린 coverslipped 슬라이드에서 얼룩 패턴을 습득하고 분석 다양한 형태로 디지털 파일을 대상 수있는의 추가한 이점을 제공합니다. 우리 세 항체의 예제를 제공하기는하지만,이 절차 특히 다른 표적 단백질뿐만 아니라 histological 얼룩, cytoarchitecture 다양한 대뇌 피질의 영역을 표시하고 따라서 매핑 목적으로 사용되는 이들의 다양한 적용할 수 있습니다.

<table><tbody><tr><td>anti-FMRP monoclonal antibody</td><td>Chemicon International</td><td>MAB2160</td><td>Requires antigen retrieval in fixed tissue.</td></tr><tr><td>anti-NeuN monoclonal antibody</td><td>Chemicon International</td><td>MAB377</td><td>N/A</td></tr><tr><td>anti-Neurofilament H monoclonal antibody</td><td>Sternberger Monoclonals Inc.</td><td>SMI32</td><td>N/A</td></tr><tr><td>Antigen Unmasking Solution</td><td>Vector Laboratories</td><td>H-3300</td><td>N/A</td></tr><tr><td>Normal horse serum</td><td>Invitrogen</td><td>16050122</td><td>500 mL</td></tr><tr><td>Biotinylated Anti-Mouse IgG (H+L), made in horse</td><td>Vector Laboratories</td><td>BA-2000</td><td></td></tr><tr><td>VECTASTAIN ABC Kit (Standard)</td><td>Vector Laboratories</td><td>PK-4000</td><td>1.5 mg</td></tr><tr><td>Staining dish for floating sections</td><td>HistoTools</td><td>10009</td><td>12 x 65 mm</td></tr><tr><td>Staining transport basket</td><td>HistoTools</td><td>10012</td><td>large&lt;br/&gt;(fits 12 x 65 mm dish)</td></tr><tr><td>Triton X-100</td><td>Fisher Scientific</td><td>P8514B</td><td>N/A</td></tr><tr><td>DAB</td><td>Fisher Scientific</td><td>AC11209-0050</td><td>N/A</td></tr></tbody></table>

batch immunostaining for large-scale protein detection in the whole monkey brain

Immunohistochemistry (IHC)는 대상 단백질의 검출에 가장 널리 사용되는 실험 기법 중 하나입니다<em&gt; 현장에서</em&gt;. 전체 두뇌를 통해 대상 단백질의 표현 패턴에 관한 질문은 설치류의 것과 작은 두뇌에 IHC를 사용하는 경우 답변을 상대적으로 쉽습니다. 그러나, 영장류의 것과 같은 대규모 뒤얽힌 두뇌에서 같은 질문에 대답하는 것은 도전의 번호를 보여줍니다. 여기서 우리는 어른 원숭이 두뇌의 대상 단백질의 면역을위한 체계적인 접근법을 제시한다. 이 방법은 신경 과학의 방법론 어소 (NSA)뿐만 아니라 자유 부동 섹션의 배치 얼룩을 위해 특별히 개발된 도구를 포함하고 sectioning 조직에 의존합니다. 그것은 특정 간격으로, 전체 두뇌를 대표하는, 섹션의 집합의 유니폼을 얼룩을 초래합니다. 그 결과 얼룩진 부분은 단백질 수준, 특정 단백질을 표현 뉴런의 인구를 측정하기 위해 분석 절차의 다양한 받게 수 있습니다.

Watch this Scientific Journal Video about Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain at JoVE.com

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

전체 영장류 두뇌를 통해 대상 단백질의 대규모 면역이 포함 소설 조직을 고용하고 주어진 시간에 여러 자유 부동 섹션의 배치 얼룩을위한 창조적인 장치를 이용하여 결합 방법을 sectioning에서 가능합니다.

전체 몽키 브레인에서 대규모 단백질 탐지에 대한 배치 Immunostaining

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

batch-immunostaining-for-large-scale-protein-detection-whole-monkey

Nanyang Technological University

Research

JoVE Journal

Biology

12.6K 조회수.  Montreal Neurological Institute. 전체 영장류 두뇌를 통해 대상 단백질의 대규모 면역이 포함 소설 조직을 고용하고 주어진 시간에 여러 자유 부동 섹션의 배치 얼룩을위한 창조적인 장치를 이용하여 결합 방법을 sectioning에서 가능합니다.

비디오: Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

로던트에서 뉴런과 비 인간 메이트 뇌 조각의 DiOLISTIC 라벨링

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

연구

신경과학

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

의미 심장한 복잡한 두뇌 지형에 대한 Wholemount Immunohistochemistry

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

미세 아교 세포와 신경 세포에 대한 예 염색 : 저온 절단 된 쥐의 뇌 조직에서 면역 조직 화학에 대한 입문서

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

면역 조직 화학 및 전자 현미경을 사전은 임베딩에 대한 비 인간 영장류 뇌 조직의 준비

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

마우스 피 질 세포 조직의 대형 3-차원 영상

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

마우스 뇌의 미세 교밀도, 형태학 및 말초 골수성 세포 침투 분석을 위해 IBA1 및 TMEM19를 사용한 면역형광 염색

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

면역학 및 감염병학

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

근 적외선 형광 및 고 분해능 주사를 사용 하 여 설치류 뇌의 단백질 발현 측정

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

Free-floating Immunostaining of Mouse Brains

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the regulation of energy metabolism and other neurobiological processes. However, the quality, reliability, and reproducibility of brain histology results may vary among laboratories. For each staining experiment, it is necessary to optimize the key procedures based on differences in species, tissues, targeted proteins, and the working conditions of the reagents. This paper demonstrates a reliable workflow in detail, including intra-aortic perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging, which can be followed easily by researchers in this field.
Also discussed are how to modify these procedures to satisfy the individual needs of researchers. To illustrate the reliability and efficiency of this protocol, perineuronal nets were stained with biotin-labeled Wisteria florbunda agglutinin (WFA) and arginine vasopressin (AVP) with an anti-AVP antibody in the mouse brain. Finally, the critical details for the entire procedure have been addressed, and the advantages of this protocol compared to those of other protocols. Taken together, this paper presents an optimized protocol for free-floating immunostaining of mouse brain tissue. Following this protocol makes this process easier for both junior and senior scientists to improve the quality, reliability, and reproducibility of immunostaining studies.

This protocol describes an efficient and reproducible approach for mouse brain histological studies, including perfusion, brain sectioning, free-floating immunostaining, tissue mounting, and imaging.

마우스 뇌의 자유 부동 면역 염색

Immunohistochemical staining of mouse brains is a routine technique commonly used in neuroscience to investigate central mechanisms underlying the ...

<meta charset="utf8"></head><body>마모셋 뇌의 체적 이미징 및 축삭 투영 매핑(Volumetric Imaging and Axonal Projection Mapping in the Marmoset Brain)

Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. We successfully implemented this technique for tracing the axonal projections in the marmoset brain. Here, the detailed experimental procedures that resulted in reliable volumetric imaging of the whole marmoset brain are described. A key process for successful imaging was the removal of meninges surrounding the brain, which interferes with slicing. A big advantage of this methodology is that the sliced sections can be used for additional staining. In the original set-up, the sliced sections are scrambled in the water bath. These sections can be correctly aligned in their original order according to the blood vessel patterns in the cortex. An example of effective histology is the visualization of myelin structure by simple light reflection, which can be combined with Nissl staining to define anatomical borders. These sections can also be used for immunological detection of non-fluorescent anterograde and retrograde tracers, which can be registered to the STPT data for layering of multiple data.

<meta charset="utf8"></head><body>신경 해부학적 분석을 위한 전체 마모셋 뇌의 연속 이광자 단층촬영(Serial Two-Photon Tomography of the whole Marmoset Brain)

Serial two-photon tomography (STPT) imaging is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. Here we describe a protocol for implementing it for marmoset brains to better understand their structural features.

신경 해부학적 분석을 위한 전체 마모셋 뇌의 연속 이광자 단층촬영(Serial Two-Photon Tomography of the whole Marmoset Brain)

Serial Two-photon tomography (STPT) is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic ...

전체 몽키 브레인에서 대규모 단백질 탐지에 대한 배치 Immunostaining

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면역 조직 화학 및 전자 현미경을 사전은 임베딩에 대한 비 인간 영장류 뇌 조직의 준비

마우스 피 질 세포 조직의 대형 3-차원 영상

마우스 뇌의 미세 교밀도, 형태학 및 말초 골수성 세포 침투 분석을 위해 IBA1 및 TMEM19를 사용한 면역형광 염색

근 적외선 형광 및 고 분해능 주사를 사용 하 여 설치류 뇌의 단백질 발현 측정

마우스 뇌의 자유 부동 면역 염색

신경 해부학적 분석을 위한 전체 마모셋 뇌의 연속 이광자 단층촬영(Serial Two-Photon Tomography of the whole Marmoset Brain)

신경 해부학적 분석을 위한 전체 마모셋 뇌의 연속 이광자 단층촬영(Serial Two-Photon Tomography of the whole Marmoset Brain)